U.S. patent application number 10/250595 was filed with the patent office on 2004-04-01 for process for producing d-mannitol.
Invention is credited to Airaksinen, Ulla, Weymarn, Niklas Von.
Application Number | 20040063183 10/250595 |
Document ID | / |
Family ID | 8559756 |
Filed Date | 2004-04-01 |
United States Patent
Application |
20040063183 |
Kind Code |
A1 |
Weymarn, Niklas Von ; et
al. |
April 1, 2004 |
Process for producing d-mannitol
Abstract
High concentration of free cells of heterofermentative lactic
acid bacteria (LAB) in a resting or slowly growing state are used
to convert fructose into mannitol. Efficient volumetric mannitol
productivities and mannitol yields from fructose are achieved in a
process applying cell-recycle, continuous stirred tank reactor
and/or circulation techniques with native LAB cells or with LAB
cells with inactivated fructokinase gene(s). Mannitol is recovered
in high yield and purity with the aid of evaporation and cooling
crystallization.
Inventors: |
Weymarn, Niklas Von;
(Helsinki, FI) ; Airaksinen, Ulla; (Vantaa,
FI) |
Correspondence
Address: |
MCDONNELL BOEHNEN HULBERT & BERGHOFF
300 SOUTH WACKER DRIVE
SUITE 3200
CHICAGO
IL
60606
US
|
Family ID: |
8559756 |
Appl. No.: |
10/250595 |
Filed: |
October 30, 2003 |
PCT Filed: |
December 19, 2001 |
PCT NO: |
PCT/FI01/01127 |
Current U.S.
Class: |
435/158 |
Current CPC
Class: |
C12P 7/18 20130101 |
Class at
Publication: |
435/158 |
International
Class: |
C12P 007/18 |
Foreign Application Data
Date |
Code |
Application Number |
Dec 20, 2000 |
FI |
20002792 |
Claims
1. A process for the production of mannitol by bioconversion,
comprising the steps of bringing a high initial concentration of
free, mannitol-producing lactic acid bacterial cells into contact
with a low-nutrient medium supplemented with a substrate
convertible into mannitol, and a cosubstrate, in a bioreactor
system, performing the bioconversion under conditions suitable for
converting said substrate into mannitol, separating the bacterial
cells from the medium by filtration to obtain a cell-free solution,
recovering from the cell-free solution the mannitol produced, and
reusing the separated bacterial cells in the bioreactor system.
2. The process according to claim 1, wherein the bacterial cells
are native lactic acid bacterial cells.
3. The process according to claim 2, wherein the bacterial cells
are of the strain Leuconostoc mesenteroides ATCC-9135.
4. The process according to claim 1, wherein the bacterial cells
are fructokinase-inactivated lactic acid bacterial cells.
5. The process according to claim 4, wherein the cells are of the
strain Leuconostoc pseudomesenteroides BPT143 (DSM 14613).
6. The process according to claim 1, wherein the substrate is
fructose.
7. The process according to claim 1, wherein the cosubstrate is
glucose.
8. The process according to claim 1, wherein the bioconversion is
performed until at least 70%, preferably 90% or more of the said
substrate has been consumed.
9. The process according to claim 1, wherein an average volumetric
mannitol productivity of at least 10 g/L/h is achieved.
10. The process according to claim 1, wherein the bioconversion is
performed as a batch process in a bioreactor system comprising a
bioconversion reactor unit and a filtration unit.
11. The process according to claim 10, comprising the steps of
separating the bacterial cells from the medium after the
bioconversion step, and reusing said cells in successive
bioconversions.
12. The process according to claim 1, wherein the bioconversion is
performed as a circulation process in a bioreactor system
comprising a bioconversion reactor unit, a filtration unit and a
mixing reactor unit.
13. The process according to claim 12, wherein the bacterial cells
are circulated between the bioconversion reactor unit and the
filtration unit.
14. The process according to claim 12 or 13, comprising the steps
of leading the cell-free solution obtained by the filtration into
the mixing reactor, and transferring the solution from the mixing
reactor back to the bioconversion reactor unit.
15. The process according to any one of the claims 12 to 14,
comprising emptying the mixing reactor after the bioconversion
step, and re-filling it with said low-nutrient medium supplemented
with said substrate and co-substrate, to run successive
bio-conversions reusing said bacterial cells.
16. The process according to claim 1, wherein the bioconversion is
performed as a continuous process in a bioreactor system comprising
a mixing tank, a feed tank, a bioconversion reactor unit, a
filtration unit and a recovery tank.
17. The process according to claim 16, wherein the bacterial cells
are circulated between the bioconversion reactor unit and the
filtration unit.
18. The process according to claim 16 or 17, comprising the steps
of feeding the bioconversion reactor unit continuously with said
low-nutrient medium supplemented with said substrate and
co-substrate, and removing the cell-free solution gained by
filtration from the bioreactor to withhold a constant volume in the
bioconversion reactor unit.
19. The process according to any one of the claims 10 to 18,
wherein the filtration unit is a tangential flow filtration
unit.
20. The process according to any one of the claims 10 to 18,
wherein said bioconversion is run in series or in parallel.
21. A bacterial strain of the genus Lactobacillus or Leuconostoc,
in which the fructokinase enzyme(s) is/are inactivated.
22. The bacterial strain according to claim 21, in which the
fructokinase enzyme(s) is/are inactivated by random
mutagenesis.
23. The bacterial strain according to claim 22, which is
Leuconostoc pseudomesenteroides BPT143 (DSM 14613).
24. The bacterial strain according to claim 21, in which the
fructokinase enzyme(s) is/are inactivated by directed
mutagenesis.
25. Use of a bacterial strain of the genus Lactobacillus or
Leuconostoc, in which the fructokinase enzyme(s) is/are
inactivated, for producing mannitol.
Description
FIELD OF THE INVENTION
[0001] This invention relates to the use of microorganisms, namely
lactic acid bacteria (LAB), and concerns particularly a new process
for the bioconversion of fructose into mannitol with free, native
or fructokinase inactivated cells in a resting or a slowly growing
state. The invention also relates to the re-use of the cell biomass
for successive bioconversions.
BACKGROUND OF THE INVENTION
[0002] D-mannitol is a six-carbon sugar alcohol, which is about
half as sweet as sucrose. It is found in small quantities in most
fruits and vegetables (Ikawa et al., 1972; Br, 1985). Mannitol is
widely used in various industrial applications. The largest
application of mannitol is as a food additive (E421), where it is
used e.g. as a sweet tasting bodying and texturing agent (Soetaert
et al., 1999). Crystalline mannitol is non-sticky, i.e. it prevents
moisture absorption, and is therefore useful as coating material of
e.g. chewing gums and pharmaceuticals. In medicine, mannitol is
used as osmotic diuretic for intoxication therapy and in surgery,
parenteral mannitol solutions are applied to prevent kidney failure
(Soetaert et al., 1999). Mannitol is also used in brain surgery to
reduce cerebral edema.
[0003] At present, commercial production of mannitol is done by
catalytic hydrogenation of invert sugar with the co-production of
another sugar alcohol, sorbitol. Typically, the hydrogenation of a
50/50-fructose/glucose mixture results in a 30/70 mixture of
mannitol and sorbitol (Soetaert et al., 1999). Besides the fact
that mannitol is the by-product of the chemical production process
and thus liable to supply problems, it is also relatively difficult
to separate from sorbitol. In contrast to most sugars and other
sugar alcohols mannitol dissolves poorly in water (13% (w/w) at
14.degree. C. (Perry et al., 1997)). Cooling crystallization is
therefore commonly used as a separation method for mannitol.
However, according to Takemura et al. (1978) the yield of
crystalline mannitol in the chemical process is still only
approximately 17% (w/w) based on the initial sugar substrates.
[0004] In order to improve the total yield of mannitol it would be
advantageous to develop a process with mannitol as the main product
and with no sorbitol formation. Some alternative processes based on
the use of microbes have been suggested in the literature. Yeast,
fungi, and LAB especially, are able to effectively produce mannitol
without co-formation of sorbitol (Itoh et al. 1992). Among LAB only
heterofermentative species are known to convert fructose into
mannitol (Pilone et al. 1991; Axelsson, 1993; Soetaert et al.
1999). Species belonging to the genera Leuconostoc, Oenococcus and
Lactobacillus particularly, have been reported to produce mannitol
effectively. In addition to mannitol these microbes co-produce
lactic and acetic acid, carbon dioxide and ethanol. These
by-products are, however, easily separable from mannitol.
[0005] Soetaert and co-workers have studied the bioconversion of
fructose into mannitol with free cells of Leuconostoc
pseudomesenteroides ATCC-12291 (Soetaert et al., 1994). Using a
fed-batch cultivation protocol they reached a maximum volumetric
productivity of 11 g mannitol/L/h and a conversion efficiency of
approximately 94 mole-%. Recently, Korakli et al. (2000) reported a
100% conversion efficiency with Lactobacillus sanfranciscensis
LTH-2590. Other heterofermentative LAB reported to be good
producers of mannitol include Leuconostoc mesenteroides, Oenococcus
oeni, Lactobacillus brevis, Lactobacillus buchneri and
Lactobacillus fermentum (Pimentel et al., 1994; Salou et al. 1994;
Erten, 1998; Soetaert et al. 1999).
[0006] In JP62239995, Hideyuki et al. (1987) used free cells of Lb.
brevis. The volumetric mannitol productivity achieved in batch
fermentation was 2.4 g/L/h.
[0007] EP0486024 and EP0683152 describe a strain named Lb. sp. B001
with volumetric mannitol productivities of 6.4 g/L/h in a free cell
batch fermentation (Itoh et al, 1992; Itoh et al., 1995).
[0008] More recently, Ojamo et al. (2000) have submitted a patent
application for a process for the production of mannitol by
immobilized LAB. In this process the average volumetric mannitol
productivity and conversion efficiency achieved were approximately
20 g/L/h and 85%, respectively. A low-nutrient medium was used
which considerably lowers the production costs. Immobilization also
enables the re-use of cell biomass for successive batch
fermentations.
[0009] These inventions have not yet replaced the conventional
hydrogenation process. The free cell bioconversion processes
described to date are not entirely suitable for industrial scale
production. Volumetric productivities in the range of 20 g/L/h, as
achieved with the immobilization process, should however, be
adequate for profitable production. In order to further develop the
features of the bioconversion alternative, factors such as
equipment investment costs, robustness of the process, medium
composition (raw material costs), and mannitol yields must be
considered and improved. The goal of the present invention is to
overcome the prior disadvantages, such as the low productivities
obtained with the free cell bioconversion systems and the low
mannitol yields characteristic for all available bioconversion
systems. Thus, the goal of the present invention is to develop a
bioconversion process, which is feasible both technically and
economically.
SUMMARY OF THE INVENTION
[0010] The present invention is accomplished to overcome the
disadvantages mentioned above. The present invention provides a
process in which a high concentration of free cells of lactic acid
bacteria is applied to the bioconversion of fructose into mannitol.
During the bioconversion phase the cells are kept in a resting or a
slowly growing state by supplementing to the fructose containing
solution only minimal amounts of complex nutrients required for
growth. The present invention describes the use of an efficient,
high-yield mannitol-producing strain in the process. The strain in
question was identified by comparing the mannitol production
capabilities of different LAB species kept in a resting or slowly
growing state. The present invention also provides an efficient,
robust production process with productivities over 20 g
mannitol/L/h. In addition, the process concept described here is
simple to apply in industrial scale, and because of the
low-nutrient medium used in it, the raw material costs are
minimized. Furthermore, by inactivating the fructokinase gene a
100% yield of mannitol from fructose is obtained.
[0011] The invention thus concerns a process for the production of
mannitol by bioconversion, which process comprises the steps of
bringing a high initial concentration of free, mannitol-producing
lactic acid bacterial cells into contact with a low-nutrient medium
supplemented with a substrate convertible into mannitol, and a
cosubstrate, in a bioreactor system; performing the bioconversion
under conditions suitable for converting said substrate into
mannitol; separating the bacterial cells from the medium by
filtration to obtain a cell-free solution; recovering from the
cell-free solution the mannitol produced; and reusing the separated
bacterial cells in the bioreactor system.
[0012] Consequently, an object of the present invention is to
provide a semi-continuous or a continuous process for the
production of mannitol. One process alternative to accomplish this
is the re-use of free cell biomass in successive batch
bioconversions as shown in FIG. 1. When the initial fructose is
depleted the cells are concentrated e.g. by tangential flow
filtration (TFF), whereby the mannitol is removed from the
bioconversion reactor in the cell-free permeate. The cell
concentrate is then diluted with fresh fructose-rich solution and a
new batch is started. During the bioconversion the cells are kept
in a resting or slowly growing state.
[0013] Another embodiment of the present invention provides a
process where a fructose-rich solution in a mixing reactor is
circulated through a bioconversion reactor containing free cells in
a resting or slowly growing state. The cells are kept in the
bioconversion reactor by cell-recycle techniques (e.g. TFF; see
FIG. 2) and the cell-free permeate is re-circulated back to the
mixing reactor. The volume in the bioconversion and mixing reactors
is kept approximately constant.
[0014] A third embodiment of the present invention is a continuous
process where a fructose-rich solution is added to a bioconversion
reactor containing free cells in a resting or slowly growing state.
The cells are kept in the bioconversion reactor by cell-recycle
techniques (e.g. TFF) and the mannitol-rich, cell-free permeate is
directed to downstream processing via a recovery tank (FIG. 3). The
volume of the bioconversion reactor is kept constant by continuous
stirred tank reactor (CSTR) techniques (e.g. by level controller,
calibrated feed and harvest pumps, or balancing the bioconversion
reactor).
[0015] Furthermore, the present invention relates to the use of LAB
in the process. Several species can be used in the process with
varying yields and productivities (see Table 1 in Example 9). For
instance, Leuconostoc pseudomesenteroides has a high productivity,
but a yield less than 80%. This is due to a strong leakage of
fructose substrate to the phospho-ketolase pathway via
fructokinase-catalyzed phosphorylation. On the other hand,
Lactobacillus sanfranciscensis gives a 100% mannitol yield from
fructose, but is low in productivity (less than 0.5 g/L/h). To have
both a high productivity and to maximize the yield, the
fructokinase gene(s) is/are inactivated in the present invention in
a high productivity species like Leuconostoc mesenteroides,
Leuconostoc pseudomesenteroides or Lactobacillus fermentum.
[0016] Consequently, further objects of the invention are bacterial
strains of the genus Lactobacillus or Leuconostoc, in which the
fructokinase enzyme(s) is/are inactivated.
BRIEF DESCRIPTION OF THE DRAWINGS
[0017] FIG. 1. Batch process alternative. Phase 1: Bioconversion.
Phase 2: Product recovery and cell concentration with tangential
flow filtration (TFF). Phase 3: Addition of fresh fructose-rich
solution to the concentrated cell suspension.
[0018] FIG. 2. Circulation process alternative. The cells are kept
within the system consisting of the bioconversion reactor unit, the
retentate side of the filtration unit and the circulation loop.
Fructose-containing solution is pumped from the mixing reactor at
the same flow rate as permeate is added to the mixing reactor.
[0019] FIG. 3. Continuous process alternative. Fresh fructose-rich
solution is prepared in the mixing reactor, which is then
transferred into the feed tank. Solution is added to and removed
from the bioconversion reactor system, consisting of the
bioconversion reactor unit, the retentate side of the filtration
unit and the circulation loop, at the same flow rates.
DETAILED DESCRIPTION OF THE INVENTION
[0020] The primary embodiment of the present invention is a process
in which mannitol is produced by bioconversion from fructose with
the aid of free native or fructokinase inactivated LAB cells kept
in a resting or a slowly growing state. The volumetric mannitol
productivities and mannitol yields from fructose for such a system
are preferably above 10 g/L/h and 90 mole-%, respectively.
[0021] The preferred substrate for the bioconversion is fructose.
Sucrose can be used as well. In addition, glucose is preferred as a
co-substrate for the production of NAD(P)H, which is needed as a
cofactor in the bioconversion of fructose into mannitol. Based on a
100 mole-% bioconversion yield of fructose into mannitol, the
preferred molar ratio of fructose and glucose is 2:1. Typically
among heterofermentative lactic acid bacteria a varying fraction of
fructose that has been transported into the cells is phosphorylated
by fructokinase-catalysis to form fructose-6-P and thus, channeled
into the phosphoketolase pathway. The "leaking" fructose carbon
skeleton is then converted stepwise into end products such as
acetic and lactic acid, ethanol and carbon dioxide. When fructose
is leaking to the phosphoketolase pathway and when the mannitol
yield from fructose is less than 100 mole-%, it is preferable to
increase the fructose to glucose ratio to avoid residual glucose
concentrations. Preferred initial concentrations of fructose and
glucose vary from 50 to 200 g/L and 20 to 100 g/L, respectively.
The upper limit of initial fructose concentration is usually set by
the maximum solubility of mannitol at the bioconversion conditions
in question. An end concentration of mannitol over the maximum
solubility would result in crystalline mannitol to form in the
bioconversion reactor, which preferably should be avoided.
[0022] Instead of using high-purity fructose and/or glucose as the
substrates, also respective compounds with a lower purity can be
used as the substrate for the cells. This is preferred in order to
lower the raw material costs, which are strongly influenced by the
price of fructose and glucose. Besides the sugars noted earlier,
the bioconversion medium also needs to be supplemented at least
with complex nitrogen sources, magnesium and manganese ions. The
preferred complex nitrogen sources are yeast extract, preferably in
initial concentrations of 0.1 to 1 g/L, and tryptone, preferably in
initial concentrations of 0.2 to 2 g/L. The concentrations of
magnesium and manganese ions are preferably in the range from 0.1
to 0.5 g/L and 0.01 to 0.1 g/L, respectively. Concentrations
providing optimum mannitol production depend on the strain in
question and can therefore, deviate from the numbers shown above.
The magnesium and manganese ions can preferably be added in the
form of respective sulphates. Alternative and less expensive
complex nitrogen sources are e.g. soybean and cottonseed meal, corn
steep liquor (CSL), yeast hydrolysates etc.
[0023] The preferred minimum concentration of free cells in the
bioconversion reactor is 5 g dry cell weight/L. A value over 10 g/L
is preferred. The initial cell biomass production, which enables
the first bioconversion cycle to proceed, can be achieved by
cultivating the cells in a nutrient-rich growth medium, applying
techniques such as batch, fed-batch, or CSTR cell-recycling. The
cells are then concentrated to high cell densities, preferably 25
to 100 g dry cell weight/L, by e.g. tangential flow filtration
(TFF) or centrifugation. Once the cells are in the bioconversion
reactor, in the preferred concentrations mentioned above, the same
cells can be used for several successive batch bioconversions (see
FIGS. 1 and 2). Hence, the processes according to alternatives
shown in FIGS. 1 and 2 of the present invention are
semi-continuous.
[0024] The bioconversion and the mixing reactors are preferably
agitated vessels with the possibility to measure and control
on-line the temperature and pH of the bioconversion medium.
Pressure indicators should preferably also be available. The carbon
dioxide formed during the bioconversion is preferably released via
the headspace either in the bioconversion or in the mixing reactor
or both. The vessels are preferably made out of food-grade
stainless steel material and the system should preferably be
suitable for aseptic process protocols. Several reactors may be
used in series and/or in parallel. For instance, nitrogen flushing
of the media can be used to improve the mannitol yields from
fructose and the CO.sub.2 removal from the bioreactors.
[0025] The temperature and pH of the bioconversion medium should
preferably be controlled either in both the bioconversion and the
mixing reactor or only in one of the reactors. The temperature can
be adjusted either with e.g. water or steam, whereas the pH can be
adjusted with e.g. NaOH, KOH, NH.sub.4OH, HCl or H.sub.2SO.sub.4
solutions. The temperature and pH should preferably be adjusted
within the respective optimum values in order to provide maximum
mannitol productivity.
[0026] A suitable microorganism, in its native form, should
preferably express mannitol dehydrogenase activity and produce
mannitol as its main metabolite. Among suitable microorganisms are
Leuconostoc mesenteroides, Leuconostoc pseudomesenteroides,
Lactobacillus brevis, Lactobacillus buchneri, Lactobacillus
fermentum, Lactobacillus sanfranciscensis, and Oenococcus oeni. The
preferred species is Leuconostoc mesenteroides and especially
strain ATCC-9135. The present invention is, however, not limited to
these microorganisms. The present invention also refers to
microorganisms with activities similar to those mentioned above.
Also microorganisms derived, by e.g. recombinant techniques, from
microorganisms mentioned above or from microorganisms with
activities similar to those mentioned above, may be used in the
process.
[0027] If the concentration of free cells in the bioconversion
reactor is increasing too much so that e.g. the productivity is
decreasing from a normal value, a suitable volume of the cell
suspension can be removed from the system. In the batch version of
the present invention (FIG. 1) this is preferably done before the
fresh bio conversion medium is added to the high cell density
suspension, in order to start a new batch-cycle. In the circulation
version of the present invention (FIG. 2) the removal is preferably
done while the mixing vessel is emptied after fructose depletion
and then refilled with fresh bioconversion medium. In the
continuous version of the present invention (FIG. 3) e.g. the
dilution rate and the contents of the feeding solution are used to
control the production of mannitol. If it is necessary to remove
cells from the continuous bioconversion reactor, it can be done
applying e.g. TFF techniques.
[0028] While a microfiltration membrane or a large ultrafiltration
membrane (e.g. 1000 kDa) is used in the TFF equipment for cell
separation, it is not expected that any other component would be
concentrated to harmful levels in the system, while these are most
likely removed from the bioconversion reactor with the permeate or
alternatively consumed by the cells.
[0029] The inactivation of the fructokinase activity is
accomplished either by classical mutagenesis or by targeted gene
inactivation techniques. Classical mutagenesis is done by treating
growing cells of LAB with 1-methyl-3-nitro-1-nitrosoguanidine and
selecting for bacteria, which cannot grow on fructose as the sole
carbon source. The obtained mutants are further tested for their
ability to import fructose into the cell to assure that the growth
defect on fructose is not caused by a mutation present in fructose
permease. The fructose transport is verified using radioactively
labeled fructose in the growth medium and detecting the
radioactivity in separated washed cells. Alternatively, the
transport of fructose can be indirectly confirmed by measuring the
conversion of fructose to mannitol in growth medium containing
fructose.
[0030] The targeted inactivation of the fructokinase gene is done
either by disrupting or by deleting the fructokinase gene. The
inactivation plasmids for both purposes are constructed using a
vector plasmid with temperature sensitive replication origin to
enhance the integration event to the bacterial chromosome. One
example of this kind of plasmid is pGhost4, which is a wide
host-range plasmid, capable of replicating in many Gram-positive
bacteria (Biswas et al., 1993). In the first phase the inactivation
plasmid is transferred to LAB by electroporation and transformants
are selected at a permissive temperature using antibiotic
selection. In the second phase, integration of the plasmid to the
bacterial chromosome is achieved by growing the transformants at a
non-permissive temperature to plasmid replication, using still
antibiotic selection.
[0031] In the disruption construct an internal fragment of the
fructokinase gene is cloned to the vector plasmid and integration
at the fructokinase locus will interrupt the coding sequence and
thus prevent the formation of an active fructokinase enzyme. In the
case of targeted deletion of the fructokinase gene, integration of
the deletion plasmid in the second phase does not disrupt the
coding sequence, but creates two regions of homologous sequences,
which serve as excision sites in later steps. These regions
determining the excision sites are cloned in the deletion plasmid
in a consecutive order and all DNA sequences between these regions
will be deleted when homologous recombination occurs. Also all
plasmid sequences, together with the antibiotic resistance gene,
will be removed from the bacterial chromosome. After integration of
the deletion plasmid the transformant bacteria are grown without
antibiotic and clones sensitive to antibiotic are selected and
tested for growth on fructose. The clones that cannot grow on
fructose as sole carbon source are selected. The conversion of
fructose to mannitol will be determined, and also the growth on the
same substrates, used for native LAB strains, will be tested.
[0032] Mannitol is the main bioconversion product of the present
invention. Other bioconversion products, which are dissolved in the
medium, are e.g. acetic and lactic acid, and ethanol. Most of the
carbon dioxide in the liquid medium is preferably removed from the
system as gaseous carbon dioxide through agitation and/or nitrogen
flushing of the medium. The liquid product solution is separated
from the cells by TFF, as shown in FIG. 1 (no additional cell
separation step is needed in the other two process alternatives of
the present invention). The rest of the product recovery process
comprises of the following unit operations: concentration,
crystallization, separation, drying, and homogenization.
Alternatively also other metabolites formed, besides mannitol, can
be recovered from the bioconversion medium.
[0033] The concentration of the liquid product solution is
preferably done by evaporation. The heated concentrate is then
transferred to a cooling crystallization unit, where mannitol
crystals fall out when the temperature of the solution is
decreased. Next the crystals are separated from the mother liquor
by a drum separator and the crystals thereby collected (crystals
A). The mother liquor is either added to the next recovery cycle or
re-crystallized separately (crystals B). Alternatively, the mother
liquid, if containing residual fructose, can be recycled back to
the bioconversion step. The crude crystals (A and B) are dissolved
in hot water, where after the solution is re-crystallized in a
cooling crystallization unit. After a second drum separation step
the white crystals are dried in a vacuum or under-pressure oven.
Finally, if needed, the dry crystals are homogenized by a suitable
method. According to the protocol presented above the total
mannitol recovery yield and crystal purity achieved, is preferably
50 to 100 mass-%, and 95 to 100 mass-%, respectively.
EXAMPLE 1
[0034] Production of cells for the bioconversion phases 1
[0035] A bench-top bioreactor containing 9.7 L of nutrient-rich
fermentation medium (Soetaert et al., 1999) was inoculated with 300
mL of a 16-h cell culture of Leuconostoc pseudomesenteroides
ATCC-12291 grown in an inoculation medium (Soetaert et al., 1999).
The temperature of the growth medium (10 L) was set first at
20.degree. C. and after 56 h raised to 25.degree. C. The pH was
controlled at 5.0. The solution was slowly agitated.
[0036] After about 66 hours the cultivation was stopped and the
cells recovered by tangential flow filtration (Pellicon.RTM. 2 Mini
Holder and Biomax.RTM. 1000 (V screen) membrane, Millipore Corp.,
USA). From an initial volume of 10.9 L (.about.3 g dry cell
weight/L) a 0.7-L cell concentrate (.about.47 g dry cell weight/L)
was obtained by this filtration technique. The cell-free permeate
(10.2 L) could thereafter be used for study of mannitol recovery.
The cell concentrate can be used as the initial biomass for the
processes described in Examples 3-5.
[0037] The volumetric mannitol productivity of this free cell
process was 1.7 g/L/h.
EXAMPLE 2
[0038] Production of cells for the bioconversion phases 2
[0039] A bench-top bioreactor containing 1.9 L of MRS growth medium
(40 g/L glucose) was inoculated with 100 mL of a 10-h cell culture
of Leuconostoc mesenteroides ATCC-9135 also grown in a MRS growth
medium (30 g/L glucose). The temperature and pH of the growth
medium (2 L) were set at 30.degree. C. and 6.0, respectively. The
solution was slowly agitated.
[0040] About 9.5 hours later the cells were harvested by
centrifugation. The cell pellet was then suspended in a fresh
bioconversion medium (See Examples 3-5).
EXAMPLE 3
Production of mannitol by bioconversion in a batch mode (FIG.
1)
[0041] The cell pellets obtained in Example 2 was suspended with
fresh bioconversion medium and transferred aseptically into a
bioconversion reactor. The total volume of the solution was 425 mL
and it had the following initial composition: 100 g/L fructose, 50
g/L glucose, 1 g/L tryptone, 0.5 g/L yeast extract, 2.62 g/L
K.sub.2HPO.sub.4.3H.sub.2O, 0.2 g/L MgSO.sub.4, and 0.01 g/L
MnSO.sub.4. The cell concentration during the bioconversion was
approximately 10 g dry cell weight/L.
[0042] The temperature control was set at 30.degree. C. and the pH
was controlled at 5.0 with 3 M NaOH. The solution was slowly
agitated.
[0043] After 4.5 hours of bioconversion time the cells had consumed
all of the sugars and the experiment was ended. The average
volumetric mannitol productivity for the process was 20.7 g/L/h.
The mannitol yield from fructose was 91.2 mole-%.
[0044] Furthermore, the product solution and cells can be separated
by e.g. TFF, and the cell concentrate re-used in successive batch
bioconversions according to the process description in FIG. 1.
EXAMPLE 4
[0045] Production of mannitol by bioconversion with circulation
(FIG. 2)
[0046] The experiment set up is shown in FIG. 2. The cell pellets,
obtained as described in Example 2, were suspended in fresh
bioconversion medium lacking the sugars and transferred aseptically
to the bioconversion reactor unit. The volume in the bioconversion
reactor unit was 0.4 L. A TFF unit (Pellicon.RTM. 2 Mini Holder and
Biomax.RTM. 1000 (V screen) membrane, Millipore Corp., USA) was
attached to the bioconversion bioreactor unit and the permeate flow
was lead to a mixing reactor. The mixing reactor (volume 1.0 L) was
standing on a balance and the mass of the reactor was kept constant
by circulating medium back to the bioconversion reactor unit. The
total volume of the whole system was 1.5 L and the medium had the
following initial composition: 100 g/L fructose, 50 g/L glucose, 1
g/L tryptone, 0.5 g/L yeast extract, 2.62 g/L
K.sub.2HPO.sub.4.3H.sub.2O, 0.2 g/L MgSO.sub.4, and 0.01 g/L
MnSO.sub.4. The cell concentration in the bioconversion reactor was
8.7 g dry cell weight/L.
[0047] The temperature and the pH were controlled both in the
bioconversion reactor and in the mixing reactor units. The
temperature control was set at 30.degree. C. and the pH was
controlled at 5.0 with 3 M NaOH. Mixing was applied in both
reactors.
[0048] After 9 hours of bioconversion time the cells had consumed
all of the sugars and the experiment was ended. The average
volumetric mannitol productivity for the process was 21.6 g/L/h.
The mannitol yield from fructose was 94.0 mole-%.
EXAMPLE 5
[0049] Production of mannitol by bioconversion in a continuous
reactor (FIG. 3)
[0050] The experiment set up is shown in FIG. 3. The cell pellets,
obtained as described in Example 2, were suspended in fresh
bioconversion medium lacking the sugars and transferred aseptically
to the bioconversion reactor unit. A TFF unit (Pellicon.RTM. 2 Mini
Holder and Biomax.RTM. 1000 (V screen) membrane, Millipore Corp.,
USA) was attached to the bioconversion bioreactor unit and the
permeate flow was lead to a recovery tank. The total volume in the
bioconversion reactor unit, retentate side of the filtration unit,
and in the circulation loop was 1.0 L. The bioconversion reactor
unit was standing on a balance and the mass of the reactor was kept
constant by adding fresh medium from a feed tank. The feeding
solution following initial composition: 25 g/L fructose, 12.5 g/L
glucose, 1 g/L tryptone, 0.5 g/L yeast extract, 2.62 g/L
K.sub.2PO.sub.4.3H.sub.2O, 0.2 g/L MgSO.sub.4, and 0.01 g/L
MnSO.sub.4. The cell concentration in the bioconversion reactor, at
dilution rate 0.68 1/h, was approximately 6.9 g dry cell
weight/L.
[0051] The temperature control was set at 30.degree. C. and the pH
was controlled at 5.0 with 3 M NaOH. The reactor was slowly
agitated. A volumetric mannitol productivity of 12.5 g/L/h was
achieved. The mannitol yield from fructose was 93.0 mole-%.
EXAMPLE 6
[0052] Inactivation of the gene encoding fructokinase by random
mutagenesis
[0053] Chemical mutagenesis of L. pseudomesenteroides ATCC-12291
was done using log-phase cells (OD.sub.600 1.0) grown in M17
supplemented with 1% glucose (GM17). Cells washed with 50 mM sodium
phosphate buffer, pH 7, were treated with
1-methyl-3-nitro-1-nitrosoguanidine, 0.5 mg/ml, for 40-50 min, at
room temperature, and washed three times with the buffer above.
Washed cells were incubated in GM17, for 1 hour, at 30.degree. C.,
and plated on GM17 agarose, incubated 2 days at 30.degree. C.
Colonies on GM17 plates were replica-plated on a chemically defined
medium (CDM; Anon., 2000) supplemented with either 1% glucose or 1%
fructose. After 2 days of incubation at 30.degree. C. colonies
growing on glucose, but not on fructose, were selected. Conversion
of fructose to mannitol will indicate that the fructose permease is
not affected by the mutagen. The fructokinase inactivated
production strain, which was able to convert fructose to mannitol,
was named BPT-143. The strain was deposited according to the
Budapest Treaty at the Deutsche Sammlung von Mikroorganismen und
Zeilkulturen, GmbH, Mascheroder Weg 1b, D-34124 Braunschweig,
Germany on Nov. 13, 2001 with the accession number DSM 14613.
EXAMPLE 7
[0054] Inactivation of the gene encoding fructokinase by directed
mutagenesis
[0055] Inactivation plasmid for disrupting the fructokinase gene(s)
of Lb. fermentum is constructed by joining an internal fragment of
a fructokinase gene between suitable restriction sites of pGhost4.
The ligation mixture is electroporated to Lactococcus lactis,
transformants are incubated for 1 day, at permissive temperature,
30.degree. C., using erythromycin (Em, 5 .mu.g/ml) and screened by
PCR with pGhost4-specific primers. Recombinant plasmids, containing
the internal fragment of fructokinase gene, are isolated and
electroporated to Lb. fermentum. Transformants are incubated
anaerobically, for 1 day, at 30.degree. C., and verified by PCR
with the previously mentioned primers. Clones carrying the
recombinant plasmids selected for the integration experiments are
grown over night, at 30.degree. C., in MRS growth medium
supplemented with 5 .mu.g/ml Em. These cell suspensions are used as
inoculate for new cultures grown for 5 hours at 42.degree. C. in
same medium. Then the cell suspensions are diluted 1:100 000,
plated on MRS-Em, and incubated for 2 days at 42.degree. C.
Colonies arising in the presence of Em at 42.degree. C. will have a
disruption plasmid integrated to the chromosome at the fructokinase
locus. Disruption of the fructokinase gene(s) will result in
reduced fructokinase activity of the disruption transformants
compared to the wild type Lb. fermentum grown in MRS or CDM
supplemented with different sugars (sucrose, fructose, lactulose,
maltose, galactose or ribose) and 5 .mu.g/ml Em. Disruption of the
fructokinase gene(s) is confirmed by Southern blotting of the
chromosomal DNA isolated from the clones with reduced fructokinase
activity.
[0056] Fructokinase genes are deleted using the following protocol.
Two 0.5 kb fragments amplified by PCR from Lb. fermentum
chromosome, surrounding the targeted deletion site, are ligated to
pGhost4. The ligation mixture is electroporated to L. lactis,
transformants are incubated for 1 day at permissive temperature,
30.degree. C., using erythromycin (Em, 5 .mu.g/ml) and screened by
PCR with pGhost4-specific primers. Plasmids containing the cloned
fragments are isolated and electroporated to Lb. fermentum.
Transformants are incubated anaerobically on MRS-Em plates for 1
day at 30.degree. C. and resulting colonies are verified by
pGhost4-specific primers to ensure the presence of the recombinant
plasmids and correct insert sizes. Raising the temperature as
described for the disruption plasmids will result in integration of
the recombinant plasmid to the chromosome. Sites of the integration
are confirmed by Southern blotting of chromosomal DNA isolated from
the integrant strains. The Lb. fermentum carrying an integrated
recombinant plasmid at a fructokinase locus is then grown without
Em, at 42.degree. C., for 100 generations and plated on MRS without
Em. Omission of the antibiotic will result in dissociation of the
integrated plasmid from the chromosome. Depending on the
recombination site either restoration of the wild type or deletion
of a fructokinase gene will happen. In both cases all foreign DNA
will be removed from the chromosome. Em-sensitive clones are
detected after replica plating on MRS with and without Em. Among
the Em-sensitive clones those with reduced fructokinase activity
are selected. Deletion of the fructokinase gene is confirmed by
Southern blotting the chromosomal DNA isolated from the deletion
strains.
EXAMPLE 8
[0057] Production of mannitol by L. pseudomesenteroides with
inactivated fructokinase gene (random mutagenesis)
[0058] L. pseudomesenteroides ATCC-12291 and the clone DSM 14613
(BPT 143) with inactivated fructokinase gene (see Example 6) were
tested for mannitol production in parallel experiments. The growth
medium had the following composition: 20 g/L fructose, 10 g/L
glucose, 10 g/L tryptone, 5 g/L yeast extract, 2.62 g/L
K.sub.2HPO.sub.4.3H.sub.2O, 0.4 g/L MgSO.sub.4, and 0.02 g/L
MnSO.sub.4. The temperature and pH was set at 30.degree. C. and
5.0, respectively. The bioconversion time was 8 hours. The mannitol
yields from fructose for the native strain and the clone were 73.7
mole-% and 85.7 mole-%, respectively. Also, a 25% improvement in
volumetric mannitol productivity was observed.
EXAMPLE 9
[0059] Comparison of mannitol production capacity of lactic acid
bacteria in a resting or slow-growing state
[0060] Pre-cultures of three of the most promising strains
(preliminary comparison studies not shown) were grown in MRS growth
medium. The cell suspensions were centrifuged and the cell pellets
washed in 0.2 M phosphate buffer (pH 5.8). After an additional
centrifugation separation the cell pellets were resuspended in the
same buffer. The concentrated cell suspensions (50 mL per strain)
were added to bioreactors containing 450 mL of a bioconversion
medium. After addition the composition of the solution was the
following: 20 g/L fructose, 10 g/L glucose, 0.5 g/L tryptone, 0.25
g/L yeast extract, 2.62 g/L K.sub.2HPO.sub.4.3H.sub.2O, 0.2 g/L
MgSO.sub.4, and 0.01 g/L MnSO.sub.4.
[0061] The temperature and pH of the bioconversion medium were set
at 30.degree. C. and 5.0, respectively. The bioconversion media
were slowly agitated. The key results are shown in Table 1.
1TABLE 1 The volumetric mannitol productivities (r.sub.mtol) and
mannitol yields from glucose (Y.sub.mtol/fru) after 8 hours of
bioconversion time. r.sub.mtol Y.sub.mtol/fru Strain: (g/L/h)
(mole/mole) Leuconostoc mesenteroides ATCC-9135 2.3 97.8
Leuconostoc pseudomesenteroides ATCC-12291 1.5 79.6 Lactobacillus
fermentum NRRL-1932 1.0 86.1
EXAMPLE 10
[0062] Recovery of mannitol
[0063] The cell-free permeate, described in Example 1, was
concentrated to approximately 250 g mannitol/L by evaporating with
a Rotavapor unit. The concentrate (T=35.degree. C.) was transferred
into a cooling crystallization unit and the temperature was
linearly (15 h) decreased to 5.degree. C. The solution was slowly
agitated. The crystals were separated by filtration and the mother
liquor was re-crystallized as described above.
[0064] The wet crystals from the first and the second cycle were
combined and dissolved in distilled water (T=45.degree. C.). The
mannitol concentration of the solution was approximately 300 g/L.
The solution was transferred into a cooling crystallization unit
and the temperature was linearly (15 h) decreased to 5.degree. C.
The crystals were separated by filtration and finally, the wet
crystals were dried overnight at 60.degree. C.
[0065] The recovery yield was about 55 mass-% and the purity above
99.5 mass-%. The mannitol found in the washing solution gained from
the last crystallization step can be re-used as part of the washing
solution in the next recovery cycle. Adding this hypothetical
amount of mannitol to the crystals obtained in the first recovery
cycle a final recovery yield of about 71% was achieved.
[0066] Deposited microorganisms
[0067] The following microorganism was deposited according to the
Budapest Treaty at the Deutsche Sammlung von Mikroorganismen und
Zellkulturen, GmbH, Mascheroder Weg 1b, D-34124 Braunschweig,
Germany.
2 Microorganism Accession number Deposit date Leuconostoc pseudo-
DSM 14613 Nov. 13, 2001 mesenteroides BPT-143
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